Vaccination in twin pregnancies: comparison between immunization before conception and during pregnancy

To evaluate the development of neutralizing Anti-Spike Protein IgG (Anti-S-IgG) during twin pregnancies before conception vs. during pregnancy. In this prospective study, three blood samples were collected from pregnant women and subjected to anti-S-IgG immunodiagnostics. The patient’s medical records, including vaccination and PCR test results, were collected from the hospital’s electronic database. Age-matched non-pregnant women were used as a control group. We enrolled 83 women with twin pregnancies. 49 women were vaccinated before conception, 21 women were vaccinated during pregnancy, and 13 were not vaccinated. Of the 13 women who weren’t vaccinated, three became positive during pregnancy, and all three were severely ill. By contrast, in women who were vaccinated during or before pregnancy, COVID-19 infection during pregnancy caused only mild symptoms. A ten-fold lower level of neutralizing Anti-S-IgG in the 3rd trimester was observed in healthy women who were vaccinated before conception and remained healthy until discharge from the hospital after delivery 1605 (IQR: 763–2410) compared to the healthy women who were vaccinated during pregnancy 152 AU/mL (IQR: 54–360). This difference was higher among women who were infected by COVID-19 (as verified by a positive PCR test). The third-trimester level of neutralizing Ant-S-IgG in the infected group was 4770 AU/mL (4760–6100) in infected women vaccinated before conception compared to those vaccinated during pregnancy who had 70 AU/mL (IQR: 20–170) (p < 0.001). In women vaccinated at 13–16 weeks gestation, neutralizing Anti-S-IgG at 20–22 weeks went up to 372 AU/mL (IQR: 120–1598) but rapidly dropped to 112 AU/mL (IQR: 54–357) at 28–30 weeks, (p < 0.001), a faster decline than in women vaccinated at a median 22 weeks before conception. Being infected by COVID-19 before conception was linked to having low Anti-S-IgG levels during pregnancy, whereas being infected by COVID-19 during pregnancy led to a very high response in the 3rd trimester. In twin pregnancies, significantly lower neutralizing Anti-S-IgG levels were observed in women vaccinated during pregnancy compared to those vaccinated before conception, whether infected or not infected by COVID-19. A full course of vaccination before conception is recommended. Trial registration. ClinicalTrials.gov Protocol Registration and Results System (PRS) Receipt Release Date: October 4, 2021. https://clinicaltrials.gov/ ID: NCT04595214.

The median maternal age at enrolment was 34.3 years, the median GA at enrollment was 12.3 weeks, and the median BMI was 24.9 (all values were centered on the medians).Conception was spontaneous for all MCDC and 47.3% of the DCDA (p < 0.001).There were 37.2% nulliparous, and the proportion between Jews and Arabs was as it is in the regional population (Top of Table 1).Chronic medical complications (diabetes mellitus, hyper-orhypothyroidism, etc.) were rare, and none had chronic hypertension or cardiovascular diseases.All these features correspond to the general characteristics of our Twin Clinic population 17,18 .

Pregnancy outcomes
All MCDC pregnancies were delivered by Caesarean section (CS) compared to 57.1% of the DCDA.The remainder of the DCDA twins were delivered vaginally (40.5%) or via tool-assisted delivery (Middle of Table 1).
Among the DCDA pregnancies, 50 women delivered twins (100 babies) and ten delivered singletons.This was due to spontaneous demise (2 cases), and selective reduction due to major genetic or structural malformation (8 cases (Fig. 1).Also, there were 2 cases of complete pregnancy loss, and 12 women didn't deliver at the time we concluded the study.(Fig. 1, left side).
Among MCDA twins, 6 women delivered 12 babies, two lost their pregnancy spontaneously, and 1 didn't deliver at the time of study conclusions.(Fig. 1, right side).
Altogether 122 babies were born.(Fig. 1).There were 47.5% females (71.4% in the MCDC).No significant differences were found between the birthweight of babies born in MCDA and DCDA pregnancies.Of the live newborns, there were 55.6% who had low birthweight (< 2,500 g), but only 6.6% had very low birth weight (< 1500 g).The majority of the newborns had a normal APGAR score at 1 and 5 min, the duration of NICU days was 10 days, and no newborn death after delivery (Bottom of Table 1).www.nature.com/scientificreports/

Pregnancy complications
The rate of gestational diabetes melilotus (GDM) was 21.2%, preeclampsia was 5%, and preterm delivery (delivery < 37 weeks) was 55.4%, (Middle of Table 1) all of which are within the range of known values for twins in Israel 17,18 .

Vaccination and susceptibility to COVID-19
The study included 83 patients, of which 70 were vaccinated, and 13 were not (Table 2).Of the non-vaccinated women, nine were healthy (69.2%), and four became PCR positive (30.7%).One was diagnosed before conception, and three (23.1%)contracted COVID-19 during pregnancy (Table 2).All three were admitted to the hospital in the third trimester with very severe symptoms.Luckily, they delivered healthy babies.
As the first and second vaccinations during pregnancy were at GA 15 and 19 weeks, Testing was performed at GA 12.1 (pre-vaccination), 22.4 (3 weeks after the second vaccination), and at 29 weeks (10 weeks after the second vaccination).Pre-pregnancy vaccination was performed at a median of 22.7 weeks pre-conception.Thus, testing at the 1st, 2nd, and 3rd trimesters performed at 34.8, 45.1, and 51 weeks after the second pre-conception vaccination.
According to Table 2, among the 70 women who were vaccinated, 56 (80%) were healthy.Of the 14 patients who were immunized but viral infected (PCR +), there were five were vaccinated before pregnancy and PCR + before conception (5%), seven were vaccinated before conception and PCR + during pregnancy (10%), and two were vaccinated during pregnancy and the viral infected (2.9%) (Table 2).There was no case of PCR + before pregnancy who was PCR positive during pregnancy.Altogether, vaccination appears to protect women who were pregnant with twins; if they were PCR + , their symptoms were mild and need no special admission to the hospital before delivery.

Time of vaccination
In women who were vaccinated before conception (violin plot, Fig. 3A), the level of neutralizing Anti-S-IgG doubled from the first to the second trimester (p = 0.05), and tripled from the second to the third trimester (p < 0.01), reaching a median of 1475 AU/mL (IQR: 392-3020), reflecting a sevenfold increase during pregnancy (overall, p = 0.012).Nevo L et al. demonstrated that vaccination with BNT-162b2 Pfizer/BioNTech during singleton pregnancies was followed by a third-trimester Anti-S-IgG level of 798 AU/mL (IQR: 424-1623) 19 , indicating that vaccination during pregnancy with twins compared to singleton pregnancy is generating a weaker level of Anti-S-IgG, which also decay faster.There were 56 women who were vaccinated and healthy-37 were vaccinated before conception and 19 during pregnancy (Table 2).Among healthy vaccinated before conception (median-22 weeks before conception), the neutralizing Anti-S-IgG significantly increases from 231 AU/mL (IQR: 70-707) in the 1st trimester to 1605 AU/ mL (IQR: 763-2410) in the third trimester (p < 0.015).Among women vaccinated during pregnancy (gestational week 15-19), the levels increased from 0 in the 1st trimester to 417 AU/mL (IQR: 114-985) in the 2nd trimester (3-4 weeks after vaccination), and it then decreases back to 152 AU/mL (IQR: 54-360) (Fig. 3B).This decay of neutralizing Anti-S-IgG reflects the immunosuppression state of the pregnant women.
The main result of our study was the X10 and above levels of Anti-S-IgG when vaccination was before conception compared to vaccination during pregnancy, as manifested in the 3rd trimester (Fig. 3A,B).The serological response in women vaccinated during pregnancy showed an increase from 0 in the first trimester to a median of 372 AU/mL in the second trimester, which rapidly dropped to one-third of this amount in the third trimester to a median of 112 AU/mL (IQR: 54-357) (Fig. 3A,B).In third-trimester singleton pregnancies vaccinated during pregnancy, the Anti-S-IgG level is 380 AU/mL (IQR: 65.35-1442.5),which is higher compared to twins (p < 0.005) 14,15 .It appears that during pregnancy, the immune system is suppressed, and this immunotolerance is higher in twins.
No significant differences were found between DCDA and MCDA twins for any of the tested parameters (Table 2, supplementary Fig. 1).

Viral infection and time of vaccination
While 14 vaccinated women (out of 70-20% compared to 31% among non-vaccinated),all had very mild disease.There were 5 who were vaccinated and infected before conception, and all had low level of neutralizing Anti-S-IgG throughout pregnancy.The 9 who got infected during pregnancy had a very high level of Anti-S-IgG (Fig. 4A,B).iInfection during pregnancy is accompanied by very high level of neutralizing Anyti-S-IgG to protect these women and their babies.www.nature.com/scientificreports/

Serological response in non-pregnant women
We compared our twin pregnant women neutralizing Anti-S-IgG levels to the results of the sample of nonpregnant women of a matched tested in the same period of time.The non-pregnant women were tested one and three months after the third vaccination.The one-month delay (vaccination to serological testing) among non-pregnant women was similar to the 3-4 weeks delay between vaccination and second-trimester testing of twin pregnancy immunized during pregnancy.Anti-S-IgG is 1,485 AU/mL (IQR: 174-2950) among the nonpregnant, compared to 417 AU/mL (IQR: 114-985), p < 0.03) in pregnant women, emphasizing the weakness of the immune protection during pregnancy (Figs. 3 and 5).The three-month delay is close to the delay between pregnancy vaccination and third-trimester testing (10-12 weeks).Here, the differences are even greater among the healthy nonpregnant women; Anti-S-IgG levels were 1,710 vs. 152 in the pregnant women who were vaccinated during pregnancy, showing the immunosuppressive state response of pregnancy (Figs. 3 and 5).The story for those vaccinated before conception is totally different.Although tested at 45.1-and 51-week delay www.nature.com/scientificreports/ between vaccination and testing, the third-trimester level of Anti-S-IgG is indistinguishable from the level of non-pregnant tested after three months (1605 vs. 1710).

Discussion
We see now that COVID-19 is turning into a seasonal viral infection and multiple variants as predicted by the World Health Organization (WHO) 1,20 , and at seasonal waves new cases are added every day.Hence, information related to the anti-viral vaccination and the viral infection-remains crucial.Studies have shown that maternal vaccination effectively protects pregnant women, their fetuses, and newborns from becoming infected 8,11 .Extensive studies support the importance of vaccination of gravid subjects before and during pregnancy and have confirmed its efficacy in reducing the rate of infection and protecting them from severe symptoms 21 .However, no studies have explored twin pregnancies 22 .The current study provides the first detailed evidence on the natural history of neutralizing Anti-S-IgG in twin pregnancies.
A main result of our study is that in twin pregnancies vaccinated before conception the levels of neutralizing Anti-S-IgG in the 3rd trimester is 10 × higher than in those vaccinated during pregnancy.Vaccination before conception is accompanied by a faster increase, and a slower decay of neutralizing Anti-S-IgG compared to vaccination during pregnancy.These results underscore the importance of immunization before conception.This is important since other studies have shown that the strength of the response of the neutralizing Anti-S-IgG may influence the transfer of neutralizing Anti-S-IgG to the newborn 15,16 , Pregnant women with twins have a larger placental mass that further reduces the chest cavity, creating a greater pressure on the lungs, and on the cardiovascular system.This is accompanied by higher incidence of preeclampsia, IUGR, gestational diabetes.The latter appears to be related to immunotolerance and here we see how the immune suppression impair an effective Anti-S-IgG protection 5,6,23,24 .
One explanation for the lower relative increase and the rapid decay of Anti-S-IgG in twin gravid women vaccinated during pregnancy is the potential uptake by the larger placental mass 25 .Placental uptake of Anti-S-IgG may also explain the similar Anti-S-IgG levels found in DCDA and MCDA.It remains to seen why this was apparently not a factor in cases where vaccination took place before conception 26,27,28 .www.nature.com/scientificreports/These findings also reinforce the importance of vaccination, given that the women who were immunized before conception and during pregnancy developed very mild symptoms, whereas the unvaccinated women who contracted COVID-19 had severe inflammatory and respiratory symptoms that required hospitalization.Overall, these results confirm that in twins, as in singleton pregnancies, being immunized protects pregnant women from severe cases of COVID-19.

Limitations
The study was conducted as the COVID-19 pandemic developed.Thus, our quasi-naturalistic study constraints led to variations in the time of vaccinations and contributed considerably to the individual variance in the levels of neutralizing Anti-S-IgG.Diversity was also contributed by the wave of the pandemic, which influenced patient behavior.Due to the study's timing, we could evaluate cases who were vaccinated during and before pregnancy.Twins are the minority of pregnancies (3% of the population) , and very large cohorts were hard to ascertain since the rate of both spontaneous and IVF pregnancies went down during the pandemic.However, this actually enabled us to review multiple scenarios.Since COVID-19 is still present, we feel that the clinical question regarding the time of vaccination in pregnancy, especially twin pregnancy, is important, and our study answers this important question.Another limitation is that data regarding a cell-mediated immune response that would have enhanced our understanding of the mother's immune response and immunity was not collected.Further studies are thus warranted.

Figure 1 .
Figure 1.Flow chart of enrolment to delivery.

Figure 3 .
Figure3.The level of neutralizing Anti-S-IgG (AU/mL) in the 3 trimesters as a function of the time of vaccination, (A) Violin plot of the serological differences between patient vaccinated before conception and during pregnancy in the 3 trimesters.First and second vaccinations during pregnancy were performed at a median gestational weeks (GA) 16 and 19.Testing was performed at GA 12.1weeks (pre-vaccination), 22.4 (3 weeks after the 2nd vaccination), and at 29 weeks, 10 weeks after the second vaccination.The median preconception vaccination week was 22.7.Thus, testing at the 1st, 2nd and 3rd trimesters, respectively, should be performed 34.8, 45.1, and 51 weeks after the second pre-conception vaccination.(B) Median neutralizing Anti-S-IgG (AU/mL) with inter quartile range (IQR).Statistical significance between trimesters is shown to the right according to the Kruskal-Wallis non-parametric test.a-b: different letters represent statistical significance of individual groups where a is largest, b, is lower and ab is in between a and b.The P at the bottom is the statistical significance between the groups in each trimester, and the letters to the right of the IQR are A-the largest, B-smaller, A,B-in between A and B, and C-the smallest.

BFigure 4 .Figure 5 .
Figure 4.The level of neutralizing Anti-S IgG (AU/mL) in the PCR-positive cases in the 3 trimesters as a function of the time of vaccination (A) Violin plot of Median levels of neutralizing Anti-S-IgG (AU/mL).The green group are patients who were PCR negative to COVID-19 (healthy) whether immunized or not.PCR + patients are divided according to the time of PCR positive test, whether before conception or during pregnancy.(B) Medians with interquartile range (IQR) of the above groups.The statistical significance (P) to the right of the table compares values across trimesters according to the Kruskal-Wallis non-parametric test, and the letters to the left of the values are: a-the larger, b-the smaller, a,b -in between.The P value at the bottom is comparing values at the same trimester.The letters to the right of the IQR are-A-the largest, B-smaller, and A,B-in between.

Table 1 .
Maternal APGAR-standardized score to evaluate infants shortly after birth as developed by Virginia APGAR and include in five criteria: activity (tone), pulse, grimace, appearance, and respiration.For each criterion, newborns can receive a score from 0 to 2, and integrated a score of 10.NICU-Intensive care units, GDMgestational diabetes melilotus, PE-preeclampsia.